The immune system is activated by expression of novel antigens on the surface of cancer cells, leading to the elimination of tumor cells by a process referred to as immune surveillance. Over time, additional genetic changes allow selected cells to circumvent the immune response and develop into a fully cancerous state. This escape from immune surveillance is now considered a principle hallmark of cancer. Recently, antibodies that block the T cell inhibitory receptors CTLA-4 and PD-1 have been shown to boost anti-tumor immunity, improving survival in some cancer patients. Response rates to these extraordinary therapies are low, however, and serious immune-related side effects can complicate treatment. The goal of this Phase I SBIR proposal is to identify small molecule antagonist ligands to a transcription factor, an orphan nuclear receptor that normally suppresses production of cytokines (such as IL-2 and IL-17) in effector T cells. Accordingly, T cells from mice deficient in this receptor are hyper-responsive to stimulation and the knockout mouse is unusually susceptible to induction of autoimmune disease. Importantly, these mice also exhibit powerful anti-tumor effects. To date, no valid ligands have been described for this target. However, structural analysis and biochemical characterization at Orphagen indicates that the target is likely to function like other members of the nuclear receptor family, where binding of a small molecule ligand to a conserved pocket regulates the affinity for transcriptional coregulators on the receptor surface through allosteric modulation. Next, we developed a high throughput screening (HTS) assay for this receptor based on its interaction with a transcriptional coregulator. An antagonist to this receptor, which releases the coregulator, is predicted to overcome its trans-repressive effect, lowering the threshold for T cell activation and enhancing immune activation. Thus, our long-term objectives are to identify small molecules that reverse transcriptional suppression by this target and explore whether these molecules can stimulate T cell activation and improve tumor immunity. In Specific Aim #1 we will screen diverse compound libraries using the HTS assay to identify small molecule ligands that disrupt the interaction between the orphan receptor and a peptide derived from its coregulator at a biochemical level. We will then determine, in Specific Aim #2, whether the ligands identified in Specific Aim #1 also activate gene transcription from the target receptor in a cellular assay. We also propose to increase ligand affinity (IC50 < 0.5 ?M) by purchase of analogues to initial hits. Finally, in Specific Aim #3, we will test the ability of the identified ligands to activate IL-2 and IL-17 production in effector T cells cultured in vitro. This pharmacological evaluation will enable more intensive drug discovery with SBIR Phase II support leading to testing of lead compounds in animal models cancer immunotherapy.